Pk7wgf2

Both full-length FaTHSFA2a and FaTHSFB1a cDNAs were PCR-amplified with a pair of primers with attB1/B2 sites (Table S4) and cloned into the pDONR221 vector by using Gateway BP Clonase II Enzyme Mix (Invitrogen, Carlsbad, CA, USA). Each construct was subsequently recombined as an N-terminal fusion of GFP into the Gateway destination binary vector pK7WGF2 (Functional Genomics Division of the Department of Plant Systems Biology, Gent, Belgium), yielding 35S::GFP–FaTHSFA2a and 35S::GFP–FaTHSFB1a by an attL× attR recombination reaction (Invitrogen, Carlsbad, CA, USA). These GFP fusion constructs were isolated and transformed into floral lips of orchids by bombardment transformation [71 (link)]. A Zeiss LSM 510 META laser-scanning confocal microscope using an LD C-Apochromat 409/1.1 W objective lens was subsequently used to observe the fluorescence emitted in the transformed cells, as previously described [71 (link)].

All aglycones or conjugated flavones or isoflavone substrates were bought from Dalian Meilun Biological Technology Co. Ltd. (Dalian, Liaoning Province, China). Whereas, malonyl-CoA was purchased from Sigma-Aldrich, St. Louis, MO, United States. The vector pGEM-T easy was purchased from Promega (Madison, WI, United States); the gateway vectors pDONR221, pDEST17, pB2GW7, pB7GWIWGII, and pK7WGF2 were either purchased from Invitrogen (Rockville, MD, United States), or gifts from Dr. Richard Dixon’s Lab at the Noble Foundation, OK, United States.